Promoter molecules for use in plants

ABSTRACT

The present invention relates to polynucleotide molecules for regulating gene expression in plants. In particular, the present invention relates to promoters isolated from Brassica napus that are useful for regulating gene expression of heterologous polynucleotide molecules in plants. The present invention also relates to expression constructs and transgenic plants containing the heterologous polynucleotide molecules.

This application claims the priority of provisional application Ser. No.60/613,081, filed Sep. 24, 2004, the disclosure of which is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of plant molecular biologyand plant genetic engineering and more specifically relates topolynucleotide molecules useful for the expression of transgenes inplants.

2. Description of Related Art

One of the goals of plant genetic engineering is to produce plants withagronomically desirable characteristics or traits. Advances in geneticengineering have provided the requisite tools to transform plants tocontain and express foreign genes. The technological advances in planttransformation and regeneration have enabled researchers to take anexogenous polynucleotide molecule, such as a gene from a heterologous ornative source, and incorporate that polynucleotide molecule into a plantgenome. The gene can then be expressed in a plant cell to exhibit theadded characteristic or trait. In one approach, expression of a gene ina plant cell or a plant tissue that does not normally express such agene may confer a desirable phenotypic effect. In another approach,transcription of a gene or part of a gene in an antisense orientationmay produce a desirable effect by preventing or inhibiting expression ofan endogenous gene.

For production of transgenic plants with various desiredcharacteristics, it would be advantageous to have a variety of promotersto provide gene expression such that a gene is transcribed efficientlyin the amount necessary to produce the desired effect. The commercialdevelopment of genetically improved germplasm has also advanced to thestage of introducing multiple traits into crop plants, often referred toas a gene stacking approach. In this approach, multiple genes conferringdifferent characteristics of interest can be introduced into a plant. Itis often desired when introducing multiple genes into a plant that eachgene is modulated or controlled for optimal expression, leading to arequirement for diverse regulatory elements. In light of these and otherconsiderations, it is apparent that optimal control of gene expressionand regulatory element diversity are important in plant biotechnology.

Numerous promoters, which are active in plant cells, have been describedin the literature. These include the nopaline synthase (nos) promoterand octopine synthase (ocs) promoters carried on tumor-inducing plasmidsof Agrobacterium tumefaciens and the caulimovirus promoters such as theCauliflower Mosaic Virus (CaMV) 19S or 35S promoter (U.S. Pat. No.5,352,605), CaMV 35S promoter with a duplicated enhancer (U.S. Pat. Nos.5,164,316; 5,196,525; 5,322,938; 5,359,142; and 5,424,200), and theFigwort Mosaic Virus (FMV) 35S promoter (U.S. Pat. No. 5,378,619). Thesepromoters and numerous others have been used in the creation ofconstructs for transgene expression in plants. Other useful promotersare described, for example, in U.S. Pat. Nos. 5,391,725; 5,428,147;5,447,858; 5,608,144; 5,614,399; 5,633,441; 6,232,526; and 5,633,435,all of which are incorporated herein by reference.

While previous work has provided a number of promoters useful to directtranscription in transgenic plants, there is still a need for novelpromoters with beneficial expression characteristics. In particular,there is a need for promoters that are capable of directing expressionof exogenous genes in dicotyledonous seeds. Many previously identifiedpromoters fail to provide the patterns or levels of expression requiredto fully realize the benefits of expression of selected genes intransgenic plants. There is, therefore, a need in the art of plantgenetic engineering for novel promoters for use in dicots.

SUMMARY OF THE INVENTION

The present invention relates to promoters that are described as siliquewall preferred promoters. The phrase “silique wall preferred” refers topromoters that drive expression of operably linked genes to higherlevels in the silique wall compared with any other tissue tested.Silique wall preferred promoters are useful for production of transgenicplants with desired seed traits. These include, but are not limited to,altering oil content, protein quality, cell proliferation, ormicronutrient quality.

In one embodiment the present invention provides a promoter comprising apolynucleotide sequence substantially homologous to a polynucleotidesequence selected from the group consisting of SEQ ID NOs: 7, 8, and 9,or any fragments and variants thereof that are capable of regulatingtranscription of operably linked polynucleotide molecules, e.g., havingpromoter activity. In particular embodiments, a fragment of a sequenceprovided herein is defined as comprising at least about 30, 40, 50, 75,100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 600, 750, 900, 1000,or more contiguous nucleotides of any of the promoter sequencesdescribed herein, including, for example, SEQ ID NO: SEQ ID NOs: 7, 8,and 9.

In another embodiment, the present invention provides a plant expressionconstruct comprising a promoter comprising a polynucleotide sequencesubstantially homologous to a polynucleotide sequence described herein,for example, selected from the group consisting of SEQ ID NOs: 7, 8, and9, or any fragments or variants thereof, wherein said promoter isoperably linked to a transcribable polynucleotide molecule.

In yet another embodiment, the present invention provides a transgenicplant stably transformed with a plant expression construct comprising apromoter provided by the invention. In one embodiment, the constructcomprises a polynucleotide sequence substantially homologous to apolynucleotide sequence selected from the group consisting of SEQ IDNOs: 7, 8, and 9, or any fragments, variants, or regions thereof,wherein said promoter is operably linked to a transcribablepolynucleotide molecule.

In another embodiment, the present invention provides a method of makinga vegetable oil, comprising the steps of incorporating into the genomeof a plant a promoter of the present invention operably linked to atranscribable polynucleotide molecule conferring altered oil and/orprotein content, growing the plant to produce seeds, and extracting theoil and/or protein from the seed. In yet another embodiment, theinvention provides a method for making food or feed comprising obtaininga plant of the invention and preparing the food or feed from the plantor a part thereof.

The foregoing and other aspects of the present invention will becomemore apparent from the following detailed description.

Brief Description of the Sequences

SEQ ID NO: 1 sets forth the polynucleotide sequence for theP-BN.RPC-0:1:1 promoter.

SEQ ID NO: 2 sets forth the polynucleotide sequence for the primer GSP1.

SEQ ID NO: 3 sets forth the polynucleotide sequence for the primer AP1.

SEQ ID NO: 4 sets forth the polynucleotide sequence for the primer GSP2.

SEQ ID NO: 5 sets forth the polynucleotide sequence for the primer AP2.

SEQ ID NO: 6 sets forth the polynucleotide sequence for the primer D11nco.

SEQ ID NO: 7 sets forth the polynucleotide sequence for theP-BN.SW1-0:1:2 promoter.

SEQ ID NO: 8 sets forth the polynucleotide sequence for theP-BN.SW2-0:1:2 promoter.

SEQ ID NO: 9 sets forth the polynucleotide sequence for theP-BN.SW3-0:1:2 promoter.

SEQ ID NO: 10 sets forth the polynucleotide sequence for the primer#17637.

SEQ ID NO: 11 sets forth the polynucleotide sequence for the primer#17636.

SEQ ID NO: 12 sets forth the polynucleotide sequence for the primer#17669.

SEQ ID NO: 13 sets forth the polynucleotide sequence for the primer#17668.

SEQ ID NO: 14 sets forth the polynucleotide sequence for the primer#18210.

SEQ ID NO: 15 sets forth the polynucleotide sequence for the primer#18209.

SEQ ID NO: 16 sets forth the polynucleotide sequence for the primer#18115.

SEQ ID NO: 17 sets forth the polynucleotide sequence for the primer#18167.

SEQ ID NO: 18 sets forth the polynucleotide sequence for the primer#18206.

SEQ ID NO: 19 sets forth the polynucleotide sequence for the primer#18340.

SEQ ID NO: 20 sets forth the polynucleotide sequence for the primer#19944.

SEQ ID NO: 21 sets forth the polynucleotide sequence for the primer#19945.

SEQ ID NO: 22 sets forth the polynucleotide sequence of the 891 bpfragment containing P-BN.SW3-0:1:2.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

As used herein, the phrase “polynucleotide molecule” refers to thesingle- or double-stranded DNA or RNA of genomic or synthetic origin,i.e., a polymer of deoxyribonucleotide or ribonucleotide bases,respectively, read from the 5′ (upstream) end to the 3′ (downstream)end.

As used herein, the phrase “polynucleotide sequence” refers to thesequence of a polynucleotide molecule. The nomenclature for DNA bases asset forth at 37 CFR § 1.822 is used.

Promoters

As used herein, the term “promoter” refers to a polynucleotide moleculethat in its native state is located upstream or 5′ to a translationalstart codon of an open reading frame (or protein-coding region) and thatis involved in recognition and binding of RNA polymerase II and otherproteins (trans-acting transcription factors) to initiate transcription.A “plant promoter” is a native or non-native promoter that is functionalin plant cells. Constitutive plant promoters are functional in most orall tissues of a plant throughout plant development. Any plant promotercan be used as a 5′ regulatory element for modulating expression of aparticular gene or genes operably associated thereto. When operablylinked to a transcribable polynucleotide molecule, a promoter typicallycauses the transcribable polynucleotide molecule to be transcribed in amanner that is similar to that of which the promoter is normallyassociated. Plant promoters can include promoters produced through themanipulation of known promoters to produce artificial, chimeric, orhybrid promoters. Such promoters can also combine cis-elements from oneor more promoters, for example, by adding a heterologous regulatoryelement to an active promoter with its own partial or completeregulatory elements. Thus, the design, construction, and use of chimericor hybrid promoters comprising at least one cis-element of SEQ ID NOs:7, 8, and 9 for modulating the expression of operably linkedpolynucleotide sequences is encompassed by the present invention.

As used herein, the term “cis-element” refers to a cis-actingtranscriptional regulatory element that confers an aspect of the overallcontrol of gene expression. A cis-element may function to bindtranscription factors, trans-acting protein factors that regulatetranscription. Some cis-elements bind more than one transcriptionfactor, and transcription factors may interact with different affinitieswith more than one cis-element. The promoters of the present inventiondesirably contain cis-elements that can confer or modulate geneexpression. Cis-elements can be identified by a number of techniques,including deletion analysis, i.e., deleting one or more nucleotides fromthe 5′ end or internal to a promoter; DNA binding protein analysis usingDNase I footprinting, methylation interference, electrophoresismobility-shift assays, in vivo genomic footprinting by ligation-mediatedPCR, and other conventional assays; or by DNA sequence similarityanalysis with known cis-element motifs by conventional DNA sequencecomparison methods. The fine structure of a cis-element can be furtherstudied by mutagenesis (or substitution) of one or more nucleotides orby other conventional methods. Cis-elements can be obtained by chemicalsynthesis or by isolation from promoters that include such elements, andthey can be synthesized with additional flanking nucleotides thatcontain useful restriction enzyme sites to facilitate subsequencemanipulation.

In one embodiment, the promoters of the present invention comprisemultiple cis-elements each of which confers a different aspect to theoverall control of gene expression. In a preferred embodiment,cis-elements from the polynucleotide molecules of SEQ ID NOs: 7, 8, and9, are identified using computer programs designed specifically toidentify cis-element, domains, or motifs within sequences. Cis-elementsmay either positively or negatively regulate gene expression, dependingon the conditions. The present invention therefore encompassescis-elements of the disclosed promoters.

As used herein, the phrase “substantially homologous” refers topolynucleotide molecules that generally demonstrate a substantialpercent sequence identity with the promoters provided herein. Ofparticular interest are polynucleotide molecules wherein thepolynucleotide molecules function in plants to direct transcription andhave at least about 70% sequence identity, at least about 80% sequenceidentity, at least about 90% sequence identity, or even greater sequenceidentity, specifically including about 73%, 75%, 78%, 83%, 85%, 88%,92%, 94, 95%, 96%, 97%, 98%, 99% or greater sequence identity with thepolynucleotide sequences of the promoters described herein, for example,provided in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9. Polynucleotidemolecules that are capable of regulating transcription of operablylinked transcribable polynucleotide molecules and are substantiallyhomologous to the polynucleotide sequences of the promoters providedherein are encompassed within the scope of this invention.

As used herein, the phrase “percent sequence identity” refers to thepercentage of identical nucleotides in a linear polynucleotide sequenceof a reference polynucleotide molecule (or its complementary strand) ascompared to a test polynucleotide molecule (or its complementary strand)when the two sequences are optimally aligned (with appropriatenucleotide insertions, deletions, or gaps totaling less than 20% of thereference sequence over the window of comparison). Optimal alignment ofsequences for aligning a comparison window are well known to thoseskilled in the art and may be conducted by tools such as the localhomology algorithm of Smith and Waterman, the homology alignmentalgorithm of Needleman and Wunsch, the search for similarity method ofPearson and Lipman, and preferably by computerized implementations ofthese algorithms such as GAP, BESTFIT, FASTA, and TFASTA available aspart of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, Calif.).An “identity fraction” for aligned segments of a test sequence and areference sequence is the number of identical components which areshared by the two aligned sequences divided by the total number ofcomponents in the reference sequence segment, i.e., the entire referencesequence or a smaller defined part of the reference sequence. Percentsequence identity is represented as the identity fraction times 100. Thecomparison of one or more polynucleotide sequences may be to afull-length polynucleotide sequence or a portion thereof, or to a longerpolynucleotide sequence.

As used herein, the term “homology” refers to the level of similarity orpercent identity between polynucleotide sequences in terms of percentnucleotide positional identity, i.e., sequence similarity or identity.As used herein, the term homology also refers to the concept of similarfunctional properties among different polynucleotide molecules, e.g.,promoters that have similar function may have homologous cis-elements.Polynucleotide molecules are homologous when under certain conditionsthey specifically hybridize to form a duplex molecule. Under theseconditions, referred to as stringency conditions, one polynucleotidemolecule can be used as a probe or primer to identify otherpolynucleotide molecules that share homology. The phrase “stringentconditions” is functionally defined with regard to the hybridization ofa nucleic-acid probe to a target nucleic acid (i.e., to a particularnucleic-acid sequence of interest) by the specific hybridizationprocedure discussed in Sambrook et al. (2000). Accordingly, thenucleotide sequences of the present invention may be used for theirability to selectively form duplex molecules with complementarystretches of polynucleotide molecule fragments.

Depending on the application envisioned one may desire to employ varyingconditions of hybridization to achieve varying degrees of selectivity ofprobe towards target sequence. For applications requiring highselectivity, one will typically desire to employ relatively highstringent conditions to form the hybrids, e.g., one will selectrelatively low salt and/or high temperature conditions, such as providedby about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C. toabout 70° C. A high stringent condition, for example, is to wash thehybridization filter at least twice with high-stringency wash buffer(0.2×SSC, 0.1% SDS, 65° C.). Appropriate moderate stringency conditionsthat promote DNA hybridization, for example, 6.0× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C.,are known to those skilled in the art. Additionally, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.Additionally, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or either the temperature or the salt concentration may be heldconstant while the other variable is changed. Such selective conditionstolerate little mismatch between the probe and the template or targetstrand. Detection of polynucleotide molecules via hybridization is wellknown to those of skill in the art, and the teachings of U.S. Pat. Nos.4,965,188 and 5,176,995 are exemplary of the methods of hybridizationanalyses.

Homology can also be determined by computer programs that alignpolynucleotide sequences and estimate the ability of polynucleotidemolecules to form duplex molecules under certain stringency conditions.Polynucleotide molecules from different sources that share a high degreeof homology are referred to as “homologues”.

Methods well known to one skilled in the art may be used to identifypromoters of interest having activity similar to the promoters describedherein. For example, cDNA libraries may be constructed using cells ortissues of interest and screened to identify genes having an expressionpattern similar to that of the promoters described herein. The cDNAsequence for the identified gene may then be used to isolate the gene'spromoter for further characterization. See, for example U.S. Pat. Nos.6,096,950; 5,589,583; and 5,898,096, incorporated herein by reference.Alternately, transcriptional profiling or electronic northern techniquesmay be used to identify genes having an expression pattern similar tothat of the promoters described herein. Once these genes have beenidentified, their promoters may be isolated for furthercharacterization. See, for example U.S. Pat. Nos. 6,506,565 and6,448,387, incorporated herein by reference. The electronic northerntechnique refers to a computer-based sequence analysis which allowssequences from multiple cDNA libraries to be compared electronicallybased on parameters the researcher identifies including abundance in ESTpopulations in multiple cDNA libraries, or exclusively to EST sets fromone or combinations of libraries. The transcriptional profilingtechnique is a high-throughput method used for the systematic monitoringof gene expression profiles for thousands of genes. This DNA chip-basedtechnology arrays thousands of cDNA sequences on a support surface.These arrays are simultaneously hybridized to a population of labeledcDNA probes prepared from RNA samples of different cell or tissue types,allowing direct comparative analysis of expression. This approach may beused for the isolation of regulatory sequences such as promotersassociated with those genes.

In another embodiment, the promoter disclosed herein can be modified.Those skilled in the art can create promoters that have variations inthe polynucleotide sequence. The polynucleotide sequences of thepromoters of the present invention as shown in SEQ ID NOs: 7, 8, and 9may be modified or altered to enhance their control characteristics. Onepreferred method of alteration of a polynucleotide sequence is to usePCR to modify selected nucleotides or regions of sequences. Thesemethods are well known to those of skill in the art. Sequences can bemodified, for example by insertion, deletion, or replacement of templatesequences in a PCR-based DNA modification approach. In the context ofthe present invention, a “variant” is a promoter containing changes inwhich one or more nucleotides of an original promoter is deleted, added,and/or substituted, preferably while substantially maintaining promoterfunction. For example, one or more base pairs may be deleted from the 5′or 3′ end of a promoter to produce a “truncated” promoter. One or morebase pairs can also be inserted, deleted, or substituted internally to apromoter. In the case of a promoter fragment, variant promoters caninclude changes affecting the transcription of a minimal promoter towhich it is operably linked. A minimal or basal promoter is apolynucleotide molecule that is capable of recruiting and binding thebasal transcription machinery. One example of basal transcriptionmachinery in eukaryotic cells is the RNA polymerase II complex and itsaccessory proteins. Variant promoters can be produced, for example, bystandard DNA mutagenesis techniques or by chemically synthesizing thevariant promoter or a portion thereof.

Novel chimeric promoters can be designed or engineered by a number ofmethods. Many promoters contain cis-elements that activate, enhance, ordefine the strength and/or specificity of the promoter. For examplepromoters may contain “TATA” boxes defining the site of transcriptioninitiation and other cis-elements located upstream of the transcriptioninitiation site that modulate transcription levels. For example, achimeric promoter may be produced by fusing a first promoter fragmentcontaining the activator cis-element from one promoter to a secondpromoter fragment containing the activator cis-element from anotherpromoter; the resultant chimeric promoter may cause an increase inexpression of an operably linked transcribable polynucleotide molecule.Promoters can be constructed such that promoter fragments or elementsare operably linked, for example, by placing such a fragment upstream ofa minimal promoter. The cis-elements and fragments of the presentinvention can be used for the construction of such chimeric promoters.Methods for construction of chimeric and variant promoters of thepresent invention include, but are not limited to, combining controlelements of different promoters or duplicating portions or regions of apromoter (see, for example, U.S. Pat. Nos. 4,990,607; 5,110,732; and5,097,025, all of which are herein incorporated by reference). Those ofskill in the art are familiar with the standard resource materials thatdescribe specific conditions and procedures for the construction,manipulation, and isolation of macromolecules (e.g., polynucleotidemolecules, plasmids, etc.), as well as the generation of recombinantorganisms and the screening and isolation of polynucleotide molecules.

In another embodiment, a promoter comprising the polynucleotide sequenceshown in SEQ ID NOs: 7, 8, and 9 includes any length of saidpolynucleotide sequence that is capable of regulating an operably linkedtranscribable polynucleotide molecule. For example, the promoters asdisclosed in SEQ ID NOs: 7, 8, and 9 may be truncated or portionsdeleted and still be capable of regulating transcription of an operablylinked polynucleotide molecule. In a related embodiment, a cis-elementof the disclosed promoters may confer a particular specificity such asconferring enhanced expression of operably linked polynucleotidemolecules in certain tissues and therefore is also capable of regulatingtranscription of operably linked polynucleotide molecules. Consequently,any fragments, portions, or regions of the promoters comprising thepolynucleotide sequence shown in SEQ ID NOs: 7, 8, and 9 can be used asregulatory polynucleotide molecules, including but not limited tocis-elements or motifs of the disclosed polynucleotide molecules.Substitutions, deletions, insertions, or any combination thereof can becombined to produce a final construct.

Polynucleotide Constructs

As used herein, the phrase “polynucleotide construct” refers to anyrecombinant polynucleotide molecule such as a plasmid, cosmid, virus,autonomously replicating polynucleotide molecule, phage, or linear orcircular single-stranded or double-stranded DNA or RNA polynucleotidemolecule, derived from any source, capable of genomic integration orautonomous replication, comprising a polynucleotide molecule where oneor more polynucleotide molecule has been linked in a functionallyoperative manner. The terms “polynucleotide construct” and “construct”are used interchangeably herein.

As used herein, the phrase “operably linked” refers to a firstpolynucleotide molecule, such as a promoter, connected with a secondtranscribable polynucleotide molecule, such as a gene of interest, wherethe polynucleotide molecules are so arranged that the firstpolynucleotide molecule affects the function of the secondpolynucleotide molecule. Preferably, the two polynucleotide moleculesare part of a single contiguous polynucleotide molecule and morepreferably are adjacent. For example, a promoter is operably linked to agene of interest if the promoter regulates or mediates transcription ofthe gene of interest in a cell.

As used herein, the phrase “transcribable polynucleotide molecule”refers to any polynucleotide molecule capable of being transcribed intoan RNA molecule. Methods are known for introducing constructs into acell in such a manner that the transcribable polynucleotide molecule istranscribed into a functional mRNA molecule that is translated andtherefore expressed as a protein product. Constructs may also beconstructed to be capable of expressing antisense RNA molecules, inorder to inhibit translation of a specific RNA molecule of interest. Forthe practice of the present invention, conventional compositions andmethods for preparing and using constructs and host cells are well knownto one skilled in the art, see for example, Sambrook et al.

Constructs of the present invention would typically contain a promoteroperably linked to a transcribable polynucleotide molecule operablylinked to a 3′ transcription termination polynucleotide molecule. Inaddition, constructs may include but are not limited to additionalregulatory polynucleotide molecules from the 3′-untranslated region (3′UTR) of plant genes (e.g., a 3′ UTR to increase mRNA stability of themRNA, such as the PI-II termination region of potato or the octopine ornopaline synthase 3′ termination regions). Constructs may include butare not limited to the 5′ untranslated regions (5′ UTR) of an mRNApolynucleotide molecule which can play an important role in translationinitiation and can also be a genetic component in a plant expressionconstruct. For example, non-translated 5′ leader polynucleotidemolecules derived from heat shock protein genes have been demonstratedto enhance gene expression in plants (see, for example, U.S. Pat. Nos.5,659,122 and 5,362,865; and U.S. Published Application No.2002/0192812, herein incorporated by reference). These additionalupstream and downstream regulatory polynucleotide molecules may bederived from a source that is native or heterologous with respect to theother elements present on the promoter construct.

Thus, constructs of the present invention comprise promoters such asthose provided in SEQ ID NOs: 7, 8, and 9, or modified as describedabove, operatively linked to a transcribable polynucleotide molecule soas to direct transcription of said transcribable polynucleotide moleculeat a desired level or in a desired tissue or developmental pattern uponintroduction of said construct into a plant cell. In some cases, thetranscribable polynucleotide molecule comprises a protein-coding regionof a gene, and the promoter provides for transcription of a functionalmRNA molecule that is translated and expressed as a protein product.Constructs may also be constructed for transcription of antisense RNAmolecules or other similar inhibitory RNA in order to inhibit expressionof a specific RNA molecule of interest in a target host cell.

Exemplary transcribable polynucleotide molecules for incorporation intoconstructs of the present invention include, for example, DNA moleculesor genes from a species other than the target gene species, or evengenes that originate with or are present in the same species, but areincorporated into recipient cells by genetic engineering methods ratherthan classical reproduction or breeding techniques. Exogenous gene orgenetic element is intended to refer to any gene or DNA molecule that isintroduced into a recipient cell. The type of DNA included in theexogenous DNA can include DNA that is already present in the plant cell,DNA from another plant, DNA from a different organism, or a DNAgenerated externally, such as a DNA molecule containing an antisensemessage of a gene, or a DNA molecule encoding an artificial or modifiedversion of a gene.

The promoters of the present invention can be incorporated into aconstruct using marker genes as described and tested in transientanalyses that provide an indication of gene expression in stable plantsystems. As used herein the phrase “marker gene” refers to anytranscribable polynucleotide molecule whose expression can be screenedfor or scored in some way. Methods of testing for marker gene expressionin transient assays are known to those of skill in the art. Transientexpression of marker genes has been reported using a variety of plants,tissues, and DNA delivery systems. For example, types of transientanalyses can include but are not limited to direct gene delivery viaelectroporation or particle bombardment of tissues in any transientplant assay using any plant species of interest. Such transient systemswould include but are not limited to electroporation of protoplasts froma variety of tissue sources or particle bombardment of specific tissuesof interest. The present invention encompasses the use of any transientexpression system to evaluate promoters or promoter fragments operablylinked to any transcribable polynucleotide molecules, including but notlimited to selected reporter genes, marker genes, or genes of agronomicinterest. Examples of plant tissues envisioned to test in transients viaan appropriate delivery system would include but are not limited to leafbase tissues, callus, cotyledons, roots, endosperm, embryos, floraltissue, pollen, and epidermal tissue.

Any scorable or screenable marker gene can be used in a transient assay.Preferred marker genes for transient analyses of the promoters orpromoter fragments of the present invention include a GUS gene (U.S.Pat. No. 5,599,670, herein incorporated by reference) or a GFP gene(U.S. Pat. No. 5,491,084, herein incorporated by reference). Theconstructs containing the promoters or promoter fragments operablylinked to a marker gene are delivered to the tissues and the tissues areanalyzed by the appropriate mechanism, depending on the marker. Thequantitative or qualitative analyses are used as a tool to evaluate thepotential expression profile of the promoters or promoter fragments whenoperatively linked to genes of agronomic interest in stable plants.

Thus, in one preferred embodiment, a polynucleotide molecule of thepresent invention as shown in SEQ ID NOs: 7, 8, or 9, or fragments,variants, or derivatives thereof, capable of regulating transcription,is operably linked to a transcribable polynucleotide molecule thatprovides for a selectable, screenable, or scorable marker. Markers foruse in the practice of the present invention include, but are notlimited to transcribable polynucleotide molecules encodingβ-glucuronidase (GUS), green fluorescent protein (GFP), luciferase(LUC), proteins that confer antibiotic resistance, or proteins thatconfer herbicide tolerance. Useful antibiotic resistance markers,including those encoding proteins conferring resistance to kanamycin(nptII), hygromycin B (aph IV), streptomycin or spectinomycin (aad,spec/strep), and gentamycin (aac3 and aacC4) are known in the art.Herbicides for which transgenic plant tolerance has been demonstratedand the method of the present invention can be applied, include but arenot limited to: glyphosate, glufosinate, sulfonylureas, imidazolinones,bromoxynil, delapon, cyclohezanedione, protoporphyrionogen oxidaseinhibitors, and isoxasflutole herbicides. Polynucleotide moleculesencoding proteins involved in herbicide tolerance are known in the art,and include, but are not limited to a polynucleotide molecule encoding5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase) described inU.S. Pat. Nos. 5,627,061; 5,633,435; and 6,040,497; and aroA describedin U.S. Pat. No. 5,094,945 for glyphosate tolerance; a polynucleotidemolecule encoding bromoxynil nitrilase (Bxn) described in U.S. Pat. No.4,810,648 for Bromoxynil tolerance; a polynucleotide molecule encodingphytoene desaturase (crtI) described in Misawa et al. (1993) and Misawaet al. (1994) for norflurazon tolerance; a polynucleotide moleculeencoding acetohydroxyacid synthase (AHAS, aka ALS) described inSathasiivan et al. (1990) for tolerance to sulfonylurea herbicides; andthe bar gene described in DeBlock et al. (1987) for glufosinate andbialaphos tolerance.

In one preferred embodiment, a polynucleotide molecule of the presentinvention as shown in SEQ ID NOs: 7, 8, or 9, or fragments, variants, orderivatives thereof, capable of regulating transcription, is operablylinked to a transcribable polynucleotide molecule that is a gene ofagronomic interest. As used herein, the phrase “gene of agronomicinterest” refers to a transcribable polynucleotide molecule thatincludes but is not limited to a gene that provides a desirablecharacteristic associated with plant morphology, physiology, growth anddevelopment, yield, nutritional enhancement, disease or pest resistance,or environmental or chemical tolerance. The expression of a gene ofagronomic interest is desirable in order to confer an agronomicallyimportant trait. A gene of agronomic interest that provides a beneficialagronomic trait to crop plants may be, for example, including, but notlimited to genetic elements comprising herbicide resistance (U.S. Pat.Nos. 5,633,435 and 5,463,175), increased yield (U.S. Pat. No.5,716,837), insect control (U.S. Pat. Nos. 6,063,597; 6,063,756;6,093,695; 5,942,664; and 6,110,464), fungal disease resistance (U.S.Pat. Nos. 5,516,671; 5,773,696; 6,121,436; 6,316,407; and 6,506,962),virus resistance (U.S. Pat. Nos. 5,304,730 and 6,013,864), nematoderesistance (U.S. Pat. No. 6,228,992), bacterial disease resistance (U.S.Pat. No. 5,516,671), starch production (U.S. Pat. Nos. 5,750,876 and6,476,295), modified oils production (U.S. Pat. No. 6,444,876), high oilproduction (U.S. Pat. Nos. 5,608,149 and 6,476,295), modified fatty acidcontent (U.S. Pat. No. 6,537,750), high protein production (U.S. Pat.No. 6,380,466), fruit ripening (U.S. Pat. No. 5,512,466), enhancedanimal and human nutrition (U.S. Pat. Nos. 5,985,605 and 6,171,640),biopolymers (U.S. Pat. No. 5,958,745 and U.S. Published Application No.2003/0028917), environmental stress resistance (U.S. Pat. No.6,072,103), pharmaceutical peptides (U.S. Pat. No. 6,080,560), improvedprocessing traits (U.S. Pat. No. 6,476,295), improved digestibility(U.S. Pat. No. 6,531,648) low raffinose (U.S. Pat. No. 6,166,292),industrial enzyme production (U.S. Pat. No. 5,543,576), improved flavor(U.S. Pat. No. 6,011,199), nitrogen fixation (U.S. Pat. No. 5,229,114),hybrid seed production (U.S. Pat. No. 5,689,041), and biofuel production(U.S. Pat. No. 5,998,700), the genetic elements and transgenes describedin the patents listed above are herein incorporated by reference.

Alternatively, a transcribable polynucleotide molecule can affect theabove mentioned phenotypes by encoding a non-translatable RNA moleculethat causes the targeted inhibition of expression of an endogenous gene,for example via antisense, RNAi, or cosuppression-mediated mechanisms.The RNA could also be a catalytic RNA molecule (i.e., a ribozyme)engineered to cleave a desired endogenous mRNA product. Thus, anypolynucleotide molecule that encodes a protein or mRNA that expresses aphenotype or morphology change of interest is useful for the practice ofthe present invention.

The constructs of the present invention may in one embodiment be doubleTi plasmid border DNA constructs that have the right border (RB orAGRtu.RB) and left border (LB or AGRtu.LB) regions of the Ti plasmidisolated from Agrobacterium tumefaciens comprising a T-DNA, that alongwith transfer molecules provided by the Agrobacterium cells, permits theintegration of the T-DNA into the genome of a plant cell. The constructsalso may contain the plasmid backbone DNA segments that providereplication function and antibiotic selection in bacterial cells, forexample, an E. coli origin of replication such as ori322, a broad hostrange origin of replication such as oriV or oriRi, and a coding regionfor a selectable marker such as Spec/Strp that encodes for Tn7aminoglycoside adenyltransferase (aadA) conferring resistance tospectinomycin or streptomycin, or a gentamicin (Gm, Gent) selectablemarker gene. For plant transformation, the host bacterial strain isoften Agrobacterium tumefaciens ABI, C58, or LBA4404, however, otherstrains known to those skilled in the art of plant transformation canfunction in the present invention.

Transformed Plants and Plant Cells

As used herein, the term “transformed” refers to a cell, tissue, organ,or organism into which has been introduced a foreign polynucleotidemolecule, such as a construct. Preferably, the introduced polynucleotidemolecule is integrated into the genomic DNA of the recipient cell,tissue, organ, or organism such that the introduced polynucleotidemolecule is inherited by subsequent progeny. A “transgenic” or“transformed” cell or organism also includes progeny of the cell ororganism and progeny produced from a breeding program employing such atransgenic plant as a parent in a cross and exhibiting an alteredphenotype resulting from the presence of a foreign polynucleotidemolecule. A plant transformation construct containing a promoter of thepresent invention may be introduced into plants by any planttransformation method. Methods and materials for transforming plants byintroducing a plant expression construct into a plant genome in thepractice of this invention can include any of the well-known anddemonstrated methods including electroporation as illustrated in U.S.Pat. No. 5,384,253; microprojectile bombardment as illustrated in U.S.Pat. Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861; and6,403,865; Agrobacterium-mediated transformation as illustrated in U.S.Pat. Nos. 5,635,055; 5,824,877; 5,591,616; 5,981,840; and 6,384,301; andprotoplast transformation as illustrated in U.S. Pat. No. 5,508,184, allof which are incorporated herein by reference.

Methods for specifically transforming dicots are well known to thoseskilled in the art. Transformation and plant regeneration using thesemethods have been described for a number of crops including, but notlimited to, soybean (Glycine max), Brassica sp., Arabidopsis thaliana,cotton (Gossypium hirsutum), peanut (Arachis hypogae), sunflower(Helianthus annuus), potato (Solanum tuberosum), tomato (Lycopersiconesculentum L.), and alfalfa (Medicago sativa). It is apparent to thoseof skill in the art that a number of transformation methodologies can beused and modified for production of stable transgenic plants from anynumber of target crops of interest.

In another embodiment, the present invention provides a method of makinga vegetable oil, comprising the steps of incorporating into the genomeof a plant a promoter of the present invention operably linked to atranscribable polynucleotide molecule conferring altered oil and/orprotein content, growing the plant to produce seeds, and extracting theoil and/or protein from the seed.

The transformed plants may be analyzed for the presence of the genes ofinterest and the expression level and/or profile conferred by thepromoters of the present invention. Those of skill in the art are awareof the numerous methods available for the analysis of transformedplants. For example, methods for plant analysis include, but are notlimited to Southern blots or northern blots, PCR-based approaches,biochemical analyses, phenotypic screening methods, field evaluations,and immunodiagnostic assays.

The seeds of this invention can be harvested from fertile transgenicplants and be used to grow progeny generations of transformed plants ofthis invention. The terms “seeds” and “kernels” are understood to beequivalent in meaning. In the context of the present invention, the seedrefers to the mature ovule consisting of a seed coat, embryo, aleurone,and an endosperm.

The phrase “cell proliferation” refers to cells undergoing mitotic celldivisions, such as in rapidly growing tissues.

The phrase “micronutrient content” means the amount of micronutrients,i.e., vitamins or carotenoids, within a seed expressed on a per weightbasis.

The phrase “oil content” means the concentration of the oil fraction,expressed as a weight percentage or parts per million basis, which maybe determined, for example, by low-resolution ¹H nuclear magneticresonance (NMR) (Tiwari et al., 1974) or Rubel, 1994) or near infraredtransmittance (NIT) spectroscopy (Orman et al., 1992; Patrick et al.,1997).

The phrase “protein quality” means the level of one or more essentialamino acids, whether free or incorporated in protein, namely histidine,isoleucine, leucine, lysine, methionine, cysteine, phenylalanine,tyrosine, threonine, tryptophan, and valine.

The following examples are included to demonstrate preferred embodimentsof the present invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventors to function well in thepractice of the present invention. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments that are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the present invention, therefore all matter set forth or shownin the accompanying drawings is to be interpreted as illustrative andnot in a limiting sense.

Example 1

This example describes the identification and isolation of expressedsequence tags (ESTs) from Brassica napus, using an electronic librarysubtraction method. Proprietary B. napus EST libraries were used as thetarget tissue and proprietary Arabidopsis and B. napus cDNA libraries asthe background tissue. ESTs identified as potentially being expressedonly in the silique tissue were further characterized by hybridization.

EST Library Construction

The target tissue EST Library was constructed from a variety of tissuesisolated from greenhouse grown Brassica napus (cultivar Quantum). Theisolated tissues included: silique walls at 10, 20, 30, 40, and 50 daysafter pollination (DAP), aleurone and seed coat at 25-28 DAP. Tissueswere flash frozen in liquid nitrogen and stored at −70° C. prior to RNAextraction. RNA was extracted by grinding approximately 500 mg planttissue in liquid nitrogen with a mortar and pestle. The ground tissuewas mixed with 5 ml REC.8+(Tris-HCl, pH9, 0.8 M NaCl, 10 mM EDTA, 0.5%β-mercaptoethanol, and 0.5% cetyltrimethyl-ammonium bromide) plus 0.1 gof polyvinylpolypyrrolidone. The mixture was homogenized with a mortarand pestle and was centrifuged at 5000 g for 5 minutes. The aqueousphase was extracted once with 1.5 ml phenol:chloroform (1:1, v/v) andonce with 1.5 ml chloroform. RNA was precipitated from the aqueous layerwith an equal volume of 95% ethanol and pelleted at 10,000×g for 20minutes. Precipitated RNA was resuspended in water and one volume of 8MLiCl were added. The mixture was incubated at −20° C. for 1 hour,followed by centrifugation at 10,000×g for 20 minutes. Precipitated RNAwas washed with 70% ethanol, air dried, and dissolved in water treatedwith diethylpyrocarbonate (DEPC water). Approximately 25 μg of total RNAwas used to prepare double-stranded cDNA using the SuperScript PlasmidSystem (Invitrogen Corporation Carlsbad, Calif.) according to themanufacturer's protocol.

EST sequences were generated for each of the six libraries describedabove. Candidate sequences were identified by using the EST sequencesfrom each library as a query against Monsanto proprietary Arabidopsisand Brassica napus cDNA libraries and the public GenBank® cDNAcollection. GenBank® is the NIH genetic sequence database, an annotatedcollection of all publicly available DNA sequences. The most highlyexpressed genes from each library were identified by determining thefrequency of appearance in all cDNA collections. An additional BLASTsearch was completed to identify clones specific for each library.

A second bioinformatics screen was then done using a proprietary LibrarySubtraction Program. The six libraries described above were used astarget libraries. All available Monsanto proprietary, non-silique wallBrassica napus cDNA libraries were used as background libraries. Thesequences that were present in any of the target libraries but notpresent in any of the background libraries, were identified as meetingthe criteria for further investigation. The 200 most abundant messagesmeeting these criteria were characterized further. Fifteen sequenceswere eliminated for overlap with the initial screen. An additional sevensequences were eliminated for significant identity withretrotransposons. The remaining sequences were then BLAST searchedagainst Arabidopsis cDNA libraries derived from leaf, root, stem, flowerbud, open flower, and developing seed. Eighty-nine of these sequenceswere eliminated from consideration for having significant identity tonon-silique wall sequences. The remaining 89 EST clones were thusidentified from this screening and were further investigated byhybridization screening.

Hybridization Screening

The EST sequences identified above were digested with SalI and NotI torelease the insert. Fragments were separated on 0.8% agarose gels in1×TBE. To facilitate transfer, the gels were exposed to short wave uvlight for 2-3 minutes. The DNA was denatured in 0.5N NaOH; 1.5 M NaCland transferred to Nytran Plus (Schleicher & Schuell BioScience, Dassel,Germany) using the TurboBlotter system (Schleicher & Schuell). Blotswere hybridized to radiolabelled cDNA derived from silique walls at 10,20, 30, 40, and 50 DAP, aleurone/seed coat, developing embryo (15-18DAP), floral buds, open flower, leaf, and root using the SMART cDNA PCRamplification Kit (BD Biosciences, Franklin Lakes, N.J.) according tomanufacturer's instructions with the addition of radiolabelled dCTP tothe final PCR reaction. A number of the clones were eliminated due tohybridization to cDNAs that were derived from developing embryo (15-18DAP), floral buds, open flower, leaf, and/or root. The remaining 67clones were used as probes against SMART™ cDNAs (BD Biosciences) derivedRNA isolated from silique walls at 10, 15, 20, 25, 30, 35, 40, 45, and50 DAP, aleurone/seed coat, developing embryo (15-18 DAP), floral buds,open flower, leaf, and root. Six candidates hybridized predominantly tosilique wall and/or aleurone. One clone hybridized specifically to rootderived cDNA.

Clone LIB4156-008-R1-K1-A4 was characterized as being expressed in theroot cells. Clone LIB4153-003-R1-K9-C1 was characterized as beingexpressed in silique wall, aleurone/seed coat, and open flower. CloneLIB4156-012-R1-K1-D2 was characterized as being expressed in siliquewall, aleurone/seed coat, and root. Clone LIB4153-011-R1-K1-E3 wascharacterized as being expressed in silique wall, aleurone/seed coat,and open flower. Clone LIB4169-008-Q1-K1-G2 was characterized as beingexpressed in aleurone/seed coat. Clone LIB4169-008-Q1-K1-D8 wascharacterized as being expressed in silique wall, aleurone/seed coat,and developing embryo. Clones LIB4153-003-R1-K9-C1,LIB4156-012-R1-K1-D2, LIB4153-011-R1-K1-E3, and LIB4156-008-R1-K1-A4were submitted for sequence confirmation. All but cloneLIB4156-008-R1-K1-A4 were verified as being the expected sequence. Thecorrect clone designation for LIB4156-008-R1-K1-A4 was determined to beLIB4156-011-R1-K1-C3.

Example 2

This example describes the isolation of promoter sequences from Brassicanapus using the EST sequences identified above.

Genome Walker Library Preparation

Brassica napus leaves were harvested and kept frozen in liquid nitrogenuntil extraction. The tissue was ground to a fine powder using a mortarand pestle while keeping the tissue frozen with liquid nitrogen.Approximately 0.4 grams of ground tissue was transferred to a centrifugetube. Six milliliters of 65° C. SDS extraction buffer (1M Tris; 0.25MEDTA; pH 8.0 with HCL; 20% SDS; 5M NaCl β-mercaptoethanol (7.1:10)) wasadded just before use. The mixture was vortexed vigorously for 30-60seconds. Samples were then incubated at 65° C. for 45 minutes, withinversion every 15 minutes. Two milliliters of ice temperature 5Mpotassium acetate solution was added, the samples were inverted gentlyto mix, and then incubated on ice for 20 minutes. The samples werechloroform extracted by adding 3 mls of chloroform to each tube andshaking gently for 10 minutes. The samples were then centrifuged at9,000 RPM for 20 minutes. The supernatant was filtered through a layerof miracloth and collected in a clean centrifuge tube. The DNA wasprecipitated by adding 2 ml of 100% isopropanol gently mixed byinverting the tubes 3-4 times, and then centrifuging at 9,000 RPM for 20minutes. The isopropanol was poured off, and the pellet was resuspendedin 200 μl RNAseA solution (1 μl of a 100 mg/ml stock from Qiagen Inc.,Valencia, Calif., diluted in 1 ml 50 mM Tris-HCL; 10 mM EDTA). A volumeof 300 μl of ammonium acetate/100% isopropanol (1:7) was added. Sampleswere vortexed and spun at 9,000 RPM for 15 minutes. Resultingsupernatant was poured off, and the pellet was washed with 500 μl of 80%ethanol. The ethanol was gently decanted and the pellet was allowed toair dry on the bench top. The pellet was resuspended in approximately200 μl TE Buffer (10 mM Tris-HCL pH8, 1 mM EDTA). The resulting DNA wasused to prepare Genome Walker™ libraries following the manufacturer'srecommended protocol (Clontech, Palo Alto, Calif.).

Isolation of P-BN.RPC-0:1:1

The P-BN.RPC-0:1:1 root preferred promoter sequence [SEQ ID NO: 1] wasisolated from the Genome Walker™ libraries described above. Primers weredesigned using the manufacturer's parameters for length, meltingtemperature (Tm), and sequence composition, from the available ESTsequence of clone LIB4156-011-R1-K1-C3. A first round PCR reaction wasrun using the Genome Walker libraries as the template and the followingprimers:

GSP1 [SEQ ID NO: 2] 5′-GCTACGGCGATGAGAAGAGAAATAAAATG-3′, and AP1 [SEQ IDNO: 3] 5′-GTAATACGACTCACTATAGGGC-3′ (supplied by BD Biosciences).

The resulting amplification product was isolated using standardtechniques well know in the art. A second PCR reaction was thenperformed using the first round amplification product as the templateand the following primers:

GSP2 [SEQ ID NO: 4] 5′-CCCACAGATTTTCTATTTCGGTTCTCATAC-3′, and AP2 [SEQID NO: 5] 5′-ACTATAGGGCACGCGTGGT-3′ (supplied by BD Biosciences).

The product of the PCR reaction was purified according to standardmethodology well known in the art and cloned into pCR2.1 Topo(Invitrogen Corporation, Carlsbad, Calif.) according to manufacturer'sinstructions. The resulting plasmid was named 2d11-2. The sequence ofthis clone was determined using standard sequencing methodologies as setforth by PE Applied Biosystems, (Foster City, Calif.). To facilitatefurther cloning, an NcoI site was added to the 3′ end of the promoterfragment, following the PCR protocol recommended by the enzymemanufacturer (PE Applied Biosystems). Briefly, approximately 10nanograms of plasmid 2d11-2 was used as the template and amplified using30 nanomoles each of the following primers:

D11 nco(#18337) 5′-GAGAAACCATGGTGAATAAATGG-3′, [SEQ ID NO: 6] and AP1.[SEQ ID NO: 3, described above]

Additionally, 10 micromoles each of dATP, dCTP, dGTP, and TTP, and 2.5units of AmpliTaq Gold in 1× Opti-Prime™ Buffer 3 (Stratagene), wereused in the reaction. After an initial incubation at 95° C. for 10minutes, 25 cycles of PCR were performed with a cycle define as 92° C.for 30 seconds, 56° C. for 30 seconds, and 72° C. for 2 minutes,followed by 1 cycle of 72° C. for 7 minutes. The product of the PCRreaction was purified according to standard methodology well known inthe art and cloned into pCR2.1 Topo (Invitrogen) according tomanufacturer's instructions. The resulting plasmid was named pMON69833.The sequence of this clone was determined using standard sequencingmethodologies as set forth by PE Applied Biosystems.

Isolation of P-BN.SW1-0:1:2, P-BN.SW2-0:1:2, and P-BN.SW3-0:1:2

The silique wall preferred promoter sequences, P-BN.SW1-0:1:2,P-BN.SW2-0:1:2, and P-BN.SW3-0:1:2 were isolated from the Genome Walker™libraries using a procedure similar to that described above forP-BN.RPC-0:1:1 with the manufacturer's instruction. For the first roundPCR reactions, primers were designed for each target sequence, using themanufacturer's parameters for length, Tm and sequence composition, fromthe respective EST sequences.

The table below shows the EST clones used as template DNA and theprimers used for the first round PCR reactions in the isolation of thelisted promoter sequences.

Promoter EST clone ID Primer 1 Primer 2 P-BN.SW1-0:1:2LIB4153-003-R1-K9-C1 #17637 #17636 P-BN.SW2-0:1:2 LIB4156-012-R1-K1-D2#17669 #17668 P-BN.SW3-0:1:2 LIB4153-011-R1-K1-E3 #18210 #18209

Primer Sequences [SEQ ID NO: 10] #176375′-TTCATATCTGCGTAAGTACGTCCATGTTC-3′ [SEQ ID NO: 11] #176365′-CGAGAAACAGAATGATGAAACTAGAGAGAC-3′ [SEQ ID NO: 12] #176695′-CACCATGCTTTCACGTCTTTTGAGTTG-3′ [SEQ ID NO: 13] #176685′-TCGATGCCACGAAAATGCATAAGAAC-3′ [SEQ ID NO: 14] #182105′-CCTCATCCTCATCCTCATCCTCAACC-3′ [SEQ ID NO: 15] #182095′-CCGTACACGGTAAAATAGTTCTTGACGGA-3′

A second amplification was then performed, similar to that describedabove for P-BN.RPC-0:1:1, using the first round amplification productsfrom the respective reactions as the template, and the followingprimers:

P-BN.SW1-0:1:1 GSP2 [SEQ ID NO: 4] and AP2 [SEQ ID NO: 5] (supplied bythe manufacturer), P-BN.SW2-0:1:1 GSP1 [SEQ ID NO: 2] and AP2, [SEQ IDNO: 5] P-BN.SW3-0:1:1 GSP2 [SEQ ID NO: 4] and AP2. [SEQ ID NO: 5]

The products of the respective PCR reactions were purified according tostandard methodology well known in the art and cloned into pCR2.1 Topo(Invitrogen) according to manufacturer's instructions. The resultingplasmids were named 3d2-2, 3d3-46, and 4G5-1 for promotersP-BN.SW1-0:1:2, P-BN.SW2-0:1:2, and P-BN.SW3-0:1:2, respectively. Thesequences for these clones were determined using standard sequencingmethodologies as set forth by PE Applied Biosystems, (Foster City,Calif.).

Similar to the procedure described above for P-BN.RPC-0:1:1, an NcoIsite was added to the 3′ end of each promoter fragment to facilitatecloning. As described above, the reaction conditions for the PCRreactions followed the protocol recommended by the enzyme manufacturer(PE Applied Biosystems, Foster City, Calif.). The primers for thespecific reactions were as follows:

Plasmid DNA 3d2-2 (P-BN.SW1-0:1:2) [SEQ ID NO: 16] #181155′-CTAAAGCCATGGTCTTAGAAAAGTTG-3′, and [SEQ ID NO: 17] #181675′-AATACGACTCACTATAGGGCACGC-3′; Plasmid DNA 3d3-46 (P-BN.SW2-0:1:2) [SEQID NO: 18] #18206 5′-GAAAAGCCATGGTAAGGCCAATATT-3′, and [SEQ ID NO: 3]AP1 Plasmid DNA 4G5-1 (P-BN.SW3-0:1:2) [SEQ ID NO: 19] #183405′-CTTCTGCCATGGAAAGAAAAAGTTG-3′, and [SEQ ID NO: 3] AP1.

The products of the PCR reactions were purified according to standardmethodology well known in the art and cloned into pCR2.1 Topo(Invitrogen) according to manufacturer's instructions. The resultingplasmids were named pMON69828, pMON69829, and pMONM69830, containing thenucleic acid sequences for P-BN.SW1-0:1:2, P-BN.SW2-0:1:2, andP-BN.SW3-0:1:2, respectively. The sequences of these clones weredetermined using standard sequencing methodologies as set forth by PEApplied Biosystems.

Example 3

This example describes the construction of plant transformation vectorscontaining the promoter sequences described above. The promotersequences were linked to the marker gene, E. coli uidA, resulting inconstructs designed to demonstrate expression in Arabidopsis and Canola.

PMON69832 (P-BN.RPC-0:1:1::GUS)

A 1661 bp fragment containing P-BN.RPC-0:1:1 [SEQ ID NO: 1] was removedfrom pMON69833 by digestion with SmaI and Nco I. The fragment wasligated into the plasmid pMON65425, which had also been digested withSmaI and NcoI. The resulting plasmid, containing P-BN.RPC-0:1:1 drivingthe E. coli uidA gene and with the napin 3′ UTR was named pMON69832. Thenucleic acid sequence was determined using known methodology andconfirmed the integrity of the cloning junctions.

PMON69827 (P-BN.SW1-0:1:2::GUS)

A 1297 bp fragment containing P-BN.SW1-0:1:2 [SEQ ID NO: 7] was removedfrom pMON69828 by digestion with SmaI and Nco I. The fragment wasligated into the plasmid pMON65425, which had also been digested withSmaI and NcoI. The resulting plasmid, containing P-BN.SW1-0:1:2 drivingthe E. coli uidA gene and with the napin 3′ UTR, was named pMON69827.The nucleic acid sequence was determined using known methodology andconfirmed the integrity of the cloning junctions.

PMON69825 (P-BN.SW2-0:1:2::GUS)

A 854 bp fragment containing P-BN.SW2-0:1:2 [SEQ ID NO: 8] was removedfrom pMON69829 by digestion with SmaI and Nco I. The fragment wasligated into pMON65425, which had also been digested with SmaI and NcoI.The resulting plasmid, containing P-BN.SW2-0:1:2 driving the E. coliuidA gene and with the napin 3′ UTR was named pMON69825. The nucleicacid sequence was determined using known methodology and confirmed theintegrity of the cloning junctions.

PMON69831 (P-BN.SW3-0:1:2::GUS)

An 888 bp fragment containing P-BN.SW3-0:1:2 (SEQ ID NO: 9) was removedfrom pMON69830 by digestion with SmaI and Nco I. The fragment wasligated into pMON65425, which had also been digested with SmaI and NcoI.The resulting plasmid, containing P-BN.SW3-0:1:2 driving the E. coliuidA gene and with the napin 3′ UTR was named pMON69831. The nucleicacid sequence was determined using known methodology and confirmed theintegrity of the cloning junctions.

Example 4

This example sets forth the procedure for transforming Arabidopsisplants with the vectors described above. Arabidopsis plants were grownby sowing seeds onto 4-inch pots containing reverse osmosis water (ROW)saturated MetroMix 200 (The Scotts Company, Columbus, Ohio). The plantswere vernalized by placing the pots in a covered flat, in a growthchamber at 4-7° C., 8 hours light/day for 4-7 days. The flats weretransferred to a growth chamber at 22° C., 55% relative humidity, and 16hours light/day at an average intensity of 160-200 μEinstein/s/m². Thecover was lifted and slid back 1-inch after germination, and removedwhen the true leaves had formed. The plants were bottom watered, asneeded, with ROW until 2-3 weeks after germination. Plants were thenbottom watered, as needed, with Plantex 15-15-18 solution (PlantexCorporation Ottawa, Canada) at 50 ppm N2. Pots were thinned such that 1plant remained per pot at 2-3 weeks after germination. Once the plantsbegan to bolt, the primary inflorescence was trimmed to encourage thegrowth of axillary bolts.

Transgenic Arabidopsis thaliana plants were obtained as described byBent et al. (1994), or Bechtold et al. (1993). Cultures of Agrobacteriumtumefaciens strain ABI containing either one of the transformationvectors pMON69832, pMON69827, pMON69825, or pMON69831 were grownovernight in LB (10% bacto-tryptone, 5% yeast extract, and 10% NaCl withkanamycin (75 mg/L), chloramphenicol (25 mg/L), and spectinomycin (100mg/L)). The bacterial culture was centrifuged and resuspended in 5%sucrose+0.05% Silwet-77 solution. The aerial portions of wholeArabidopsis plants (at about 5-7 weeks of age) were immersed in theresulting solution for 2-3 seconds. The excess solution was removed byblotting the plants on paper towels. The dipped plants were placed ontheir side in a covered flat and transferred to a growth chamber at 19°C. After 16 to 24 hours the dome was removed and the plants were setupright. When plants had reached maturity, water was withheld for 2-7days prior to seed harvest. Harvested seed was passed through astainless steel mesh screen (40 holes/inch) to remove debris. Theharvested seed was then stored in paper coin envelopes at roomtemperature until analysis.

The seeds from the transgenic Arabidopsis plants were surfacedsterilized using a vapor phase sterilization protocol. Briefly, an opencontainer of seeds was placed in a dessicator with a beaker containing100 ml of household bleach. Immediately prior to sealing the dessicator,3 ml concentrated HCl was added to the bleach solution to generatechlorine gas. The dessicator is sealed and a vacuum was applied to allowsterilization by chlorine fumes. The seeds were incubated for severalhours. Sterilized seed were sprinkled onto Arabidopsis germination media[MS Salts (1×); sucrose (1%); myo-inositol (100 mg/L); thiamine-HCl (1mg/L); pyridoxine-HCl (500 mg/L); nicotinic acid (500 mg/L); MES pH 5.7(0.05%) and Phytagar (0.7%)] supplemented with 50 mg/L glyphosate. Up to16 glyphosate resistant seedlings were transplanted to 2¼-inch potscontaining MetroMix 200, thinned to one seedling per pot, and were grownunder the conditions described above until the initial siliques that hadformed began to desiccate. Tissue (rosette leaf, cauline leaf, stem,flowers, floral buds, and developing siliques) was removed from each T1plant for subsequent histochemical staining.

Canola plants were transformed using the protocol described by Moloneyand Radke in U.S. Pat. No. 5,720,871. Briefly, seeds of Brassica napusby Ebony were planted in 2-inch pots containing Metro Mix 350 (TheScotts Company, Columbus, Ohio). The plants are grown in a growthchamber at 24° C., and a 16/8 hour photoperiod, with light intensity of400 μEm-2 sec-1 (HID lamps). After 2½ weeks, the plants weretransplanted into 6-inch pots and grown in a growth chamber at 15/10° C.day/night temperature, 16/8 hour photoperiod, light intensity of 800μEm-2 sec-1 (HID lamps). Four terminal internodes from plants just priorto bolting or in the process of bolting but before flowering are removedand surface sterilized in 70% v/v ethanol for 1 minute, 2% w/v sodiumhypochlorite for 20 minutes and rinsing 3 times with sterile deionizedwater. Six to seven stem segments are cut into 5 mm discs, maintainingorientation of basal end. Cultures of Agrobacterium tumefaciens strainABI containing one of the transformation vectors pMON69832, pMON69827,pMON69825, or pMON69831 are grown overnight on a rotator shaker at 24°C. in 2 mls of Luria Broth, LB, (10% bacto-tryptone, 5% yeast extract,and 10% NaCl) containing 50 mg/l kanamycin, 24 mg/l chloramphenicol and100 mg/l spectinomycin. A 1:10 dilution is made in MS media (Murashigeand Skoog, 1962) giving approximately 9×10⁸ cells per ml. The stem discs(explants) are inoculated with 1.0 ml of Agrobacterium and the excess isaspirated from the explants. The explants are placed basal side down inpetri plates containing media comprising 1/10 MS salts, B5 vitamins (1%inositol; 0.1% thiamine HCl; 0.01% nicotinic acid; 0.01%pyridoxine-HCl), 3% sucrose, 0.8% agar, pH 5.7, 1.0 mg/l 6-benzyladenine(BA). The plates are layered with 1.5 ml of media containing MS salts,B5 vitamins, 3% sucrose, pH 5.7, 4.0 mg/l p-chlorophenoxyacetic acid,0.005 mg/l kinetin and covered with sterile filter paper. Following a 2to 3 day co-culture, the explants are transferred to deep dish petriplates containing MS salts, B5 vitamins, 3% sucrose, 0.8% agar, pH 5.7,1 mg/l BA, 500 mg/l carbenicillin, 50 mg/l cefotaxime, and 25 mg/lglyphosate for selection. Seven explants are placed on each plate. After3 weeks they are transferred to fresh media, 5 explants per plate. Theexplants are cultured in a growth room at 25° C., continuous light.

The transformed plants are grown in a growth chamber at 22° C. in a 16-8hours light-dark cycle with light intensity of 220 μEm⁻²s⁻¹ for severalweeks before transferring to the greenhouse. The plants are then grownin greenhouse conditions until maturity. The resulting mature R1 seedsare collected for analysis. Plants are maintained in a greenhouse understandard conditions. Developing seed is harvested at various stagesafter pollination and stored at negative 70° C. Mature seed is collectedand stored under controlled conditions consisting of about 17° C. and30% humidity.

Example 5

This example describes the analysis of the expression of β-glucuronidasein Arabidopsis thaliana plants transformed with pMON69832, pMON69827,pMON69825, or pMON69831 using histochemical staining. The results ofthis analysis provide a characterization of the expression for thedifferent promoters.

Freshly harvested tissues from transformed Arabidopsis plants wereincubated for approximately 24 hours at 37° C. in a solution containing50 mM NaPO₄ (pH 7.2); 100 μM potassium ferricyanide; 100 μM potassiumferrocyanide, 0.03% Triton X-100; 20% methanol and 2.5 mg/ml5-bromo-4-chloro-3-indoyl glucuronic acid (X-gluc). In some cases thepotassium ferricyanide, potassium ferrocyanide, and methanol wereomitted from the staining solution. The stained tissue was cleared ofchlorophyll by an overnight incubation in 70% ethanol/30% H₂O at 37° C.Stained tissues were photographed immediately or transferred to asolution of 70% ethanol/30% glycerol (v/v) and stored at 4° C. untilphotographed. The samples were then scored as positive (+) or negative(−) for staining a blue color.

For detection of expression in canola, up to 5 siliques were harvestedfrom individual R0 plants at several time points after pollination.Siliques were scored with an 18 gauge needle to allow the stainingsolution to contact the developing seed. The siliques were incubated forapproximately 24 hours at 37° C. in a solution containing 50 mM NaPO₄(pH 7.2); 100 μM potassium ferricyanide; 100 μM potassium ferrocyanide,0.03% Triton X-100; 20% methanol and 2.5 mg/ml 5-bromo-4-chloro-3-indoylglucuronic acid (X-gluc). The stained tissue was cleared of chlorophyllby an overnight incubation in 70% ethanol/30% H₂O at 37° C. Stainedtissues were photographed immediately or transferred to a solution of70% ethanol/30% glycerol (v/v) and stored at 4° C. until photographed.Samples were scored positive (+) or negative (−) for blue color.

For P-BN.RPC-0:1:1, a root preferred promoter contained in pMON69832,none of 10 plants screened had detectable levels of activity in thesilique wall (data not shown). For pMON69827, 3 out of 10 plantsscreened had detectable levels of activity in the silique wall from atleast one time point. For pMON69825, 6 out of 10 plants screened haddetectable levels of activity in the silique wall from at least one timepoint. For pMON69831, 5 out of 10 plants screened had detectable levelsof activity in the silique wall from at least one time point. Noactivity was detected in the seed coat or aleurone of plants transformedwith pMON69831. Time course data for individual transformants ispresented in Tables 1-3.

TABLE 1 P-BN.SW1-0:1:2 Expression in Developing Canola Siliques DaysAfter Pollination Construct Event 3 6 9 12 15 20 25 30 35 40 pMON69827BN_G1427 − − − − − − − − − − pMON69827 BN_G1428 − − − − − − − − − −pMON69827 BN_G1429 − − − − − − − − − − pMON69827 BN_G1430 − − − − − − −− − − pMON69827 BN_G1480 − − − − − − − − − − pMON69827 BN_G1481 − ND −ND + − − − − − pMON69827 BN_G1482 − − − + − + − − − + pMON69827BN_G1507 + + + + + + + + + + pMON69827 BN_G1539 − − − − − − − − − −pMON69827 BN_G1540 − − − − − − − − − − Control SP30052 − − − − − − − − −−

TABLE 2 P-BN.SW2-0:1:2 Expression in Developing Canola Siliques DaysAfter Pollination Construct Event 3 6 9 12 15 20 25 30 35 40 pMON69825BN_G1474 − − − − − − − − − − pMON69825 BN_G1475 − − − − − − − − − −pMON69825 BN_G1476 + + + + NA + + + + + pMON69825 BN_G1477 + + + NA + +− − − pMON69825 BN_G1478 − − − − − − − − − − pMON69825 BN_G1479 − ND + +− − − − − − pMON69825 BN_G1536 − − − − − − − − − + PMON69825 BN_G1537 −− − − + + + − − − PMON69825 BN_G1538 + − − − + + − − − − PMON69825BN_G1580 − − − − − − − − − − Control SP30052 − − − − − − − − − −

TABLE 3 P-BN.SW3-0:1:2 Expression in Developing canola Siliques DaysAfter Pollination Construct Event 3 6 9 12 15 20 25 30 35 40 pMON69831BN_G1839 + + + + + − + + + + pMON69831 BN_G1901 − − − − − − − − − −pMON69831 BN_G1902 − − − − − − − − − − pMON69831 BN_G1903 − − − − − − −− − − pMON69831 BN_G1904 − − − − − − − − − − pMON69831 BN_G1905 − −− + + + − − − − pMON69831 BN_G1906 − − + + + + + + + + pMON69831BN_G1907 − − − − + + + − − − pMON69831 BN_G1908 − − − − + − + − − −pMON69831 BN_G1951 − − − − − − − − − − Control SP30052 − − − − − − − − −−

The data in Tables 1-3 indicate that the three promoters, P-BN.SW1-0:1:2(SEQ ID NO: 7), P-BN.SW2-0:1:2 (SEQ ID NO: 8), and P-BN.SW3-0:1:2 (SEQID NO: 9) are capable of driving silique wall expression in Arabidopsisand Canola.

Example 6

This example describes the construction of plant transformation vectorscontaining the P-BN.SW3-0:1:2 promoter operably linked to theArabidopsis thaliana isocitrate lyase gene. To facilitate cloning, theP-BN.SW3-0:1:2 promoter was amplified using pMON69830 as a template withthe following primers:

19944 [SEQ ID NO: 20] 5′-CCCGGGCTGGTCCTGCGAAGATTCTCAG-3′, and 19945 [SEQID NO: 21] 5′-CTCGAGCGGCCGCTTCTATGTCGACTGGAAAGAAAAAGTTGTGCCAA CAAAAG-3′.

The reactions were run under conditions recommended by the Expand HighFidelity enzyme manufacturer (Roche Applied Biosciences, Indianapolis,Ind.). The product of the PCR reaction was purified according tostandard methodology well known in the art and cloned into pCR2.1 Topo(Invitrogen) according to the manufacturer's instructions. The resultingplasmid was named pMON79578.

A 910-base pair fragment containing P-Bn.SW3-0:1:2 [SEQ ID NO: 9] wasremoved from the vector pMON79578 by digestion with SmaI and NotI. Thefragment was cloned into pMON82374 which had been digested with PmeI andNotI in place of the pBr.Snap2 promoter. The vector pMON82374 contains anopaline T-DNA right border sequence and octopine T-DNA left bordersequence, with 2 expression cassettes contained between the 2 T-DNAborders. The first cassette contains the pBr.Snap2 promoter andT-Br.Snap2-1 terminator. The second cassette contains with a 35Spromoter from the Figwort Mosaic Virus (FMV) between the 2 T-DNAborders, driving the expression of a chimeric EPSP synthase genecontaining a chloroplast targeting sequence from the Arabidopsis EPSPsynthase gene (GenBank identifier number gi: 16272) linked to asynthetic EPSP synthase coding region (U.S. Pat. No. 5,633,435) and the3′ untranslated region from the pea rbcS E9 gene. Additionally,pMON82374 contains recognition sites for cre recombinase. Therecombinase sites are 5′ of the FMV promoter and 3′ of the E9 3′ UTR.The resulting plasmid was named pMON79581.

A 2111 fragment containing L-At.ICL, CR-At.ICL, and T-At.ICL was removedfrom EST clone pMON82360 by digestion with SalI and NotI restrictionenzymes and cloned between the pBr.Snap2 promoter and T-Br.Snap2-1 3′UTR in the SalI and Not I digested pMON79581. The resulting plasmid wasnamed pMON79583. The nucleic acid sequence was determined using knownmethodology and confirmed the integrity of the cloning junctions.pMON79583 was used for the transformation of Brassica napus.

An 891-base pair fragment [SEQ ID NO: 22] containing the P-Bn.SW3-0:1:2promoter sequence [SEQ ID NO: 9], was removed from the vector pMON79578by digestion with SmaI and SalI. The fragment was cloned intoDMRUEZ03.0112 which had been digested with PacI and SalI. Prior to SalIdigestion and gel purification, the PacI overhang was blunt ended usingPfu polymerase according to the manufacturer's instructions(Stratagene). The resulting plasmid contains the nopaline T-DNA rightborder sequence and octopine T-DNA left border sequence, between whichare the promoter, 5′ UTR and first intron from the Arabidopsis act7 genedriving the expression of a CP4 EPSP synthase gene. This CP4 EPSPsynthase gene comprises a CTP, linked to a synthetic EPSP synthasecoding region and the 3′ UTR from the pea rbsc E9 gene; theP-Bn.SW3-0:1:2 promoter operably linked to the Arabidopsis thalianaisocitrate lyase gene (as described in PCT Application WO 03/79766) andthe napin 3′ UTR. The nucleic acid sequence was determined using knownmethodology and confirmed the integrity of the cloning junctions. Theresulting plasmid was named pMON82378 was used for the transformation ofsoybean.

Example 7

This example sets forth the transformation of soybean plants with theconstructs described in Example 5.

Soybean plants were transformed using an Agrobacterium-mediatedtransformation method, as described by Martinell (U.S. Pat. No.6,384,301). For this method, overnight cultures of Agrobacteriumtumefaciens containing the plasmid that includes a gene of interest,such as pMON75201, were grown to log phase and then diluted to a finaloptical density of 0.3 to 0.6 using standard methods known to oneskilled in the art. These cultures were used to inoculate the soybeanembryo explants prepared as described below. Briefly, the method is adirect germline transformation into individual soybean cells in themeristem of an excised soybean embryo. The soybean embryo is removedafter surface sterilization and germination of the seed. The explantsare then plated on OR media, a standard MS medium as modified by Barwaleet al. (1986), plus 3 mg/L BAP, 200 mg/L carbenicillin, 62.5 mg/Lcefotaxime, and 60 mg/L benomyl, and stored at 15° C. overnight in thedark. The following day the explants are wounded with a scalpel bladeand inoculated with the Agrobacterium culture prepared as describedabove. The inoculated explants are then cultured for 3 days at roomtemperature. Following the post-transformation culture, the meristematicregion is then cultured on standard plant tissue culture media in thepresence of the herbicide glyphosate (Monsanto Company, St. Louis, Mo.),which acts as both a selection agent and a shoot inducing hormone. Mediacompositions and culture lengths are detailed in the aforementionedMartinell patent. After 5 to 6 weeks, the surviving explants that have apositive phenotype are transferred to soil and grown under greenhouseconditions until maturity.

The mature seeds of the transformed plants are harvested and analyzedfor oil content using standard methodology, for example by near-infraredreflectance (NIR) spectroscopy as described in Williams and Norris(1987). The seeds from plants expressing the Arabidopsis thalianaisocitrate lyase gene, driven by the P-Bn.SW3-0:1:2 promoter, have ahigher oil content as compared to seeds from non-transformed plants.

REFERENCES

The references listed below are incorporated herein by reference to theextent that they supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

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1. An isolated promoter comprising a polynucleotide sequence selectedfrom the group consisting of a) a polynucleotide sequence comprising thenucleic acid sequence of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; b) apolynucleotide sequence having at least about 70% sequence identity tothe sequence of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 that is capableof regulating transcription of an operably linked transcribablepolynucleotide molecule; and c) a fragment of the polynucleotidesequence of a) or b) capable of regulating transcription of an operablylinked transcribable polynucleotide molecule.
 2. A construct comprisingthe promoter of claim 1 operably linked to a heterologous transcribablepolynucleotide molecule.
 3. The construct of claim 2, wherein thetranscribable polynucleotide molecule is operably linked to a 3′transcription termination polynucleotide molecule.
 4. The construct ofclaim 2, wherein said transcribable polynucleotide molecule is a gene ofagronomic interest.
 5. The construct of claim 2, wherein saidtranscribable polynucleotide molecule is a marker gene.
 6. A transgenicplant stably transformed with the construct of claim
 2. 7. Thetransgenic plant of claim 6, wherein the transgenic plant is adicotyledonous plant.
 8. The transgenic plant of claim 7, wherein thedicotyledonous plant is selected from the group consisting of tobacco,tomato, potato, soybean, cotton, canola, sunflower, and alfalfa.
 9. Thetransgenic plant of claim 6, wherein the transcribable polynucleotidemolecule confers altered cell proliferation in the seed of saidtransgenic plant.
 10. The transgenic plant of claim 6, wherein thetranscribable polynucleotide molecule confers altered oil content in theseed of said transgenic plant.
 11. The transgenic plant of claim 6,wherein the transcribable polynucleotide molecule confers alteredprotein quality in the seed of said transgenic plant.
 12. The transgenicplant of claim 6, wherein said transcribable polynucleotide moleculeconfers altered micronutrient content to said transgenic plant.
 13. Aseed of the transgenic plant of claim 6, wherein the seed comprises saidconstruct.
 14. Oil of the transgenic plant of claim 6, wherein the oilcomprises a detectable nucleic acid comprising the promoter of claim 1.15. Meal of the transgenic plant of claim 6, wherein the meal comprisesa detectable nucleic acid comprising the promoter of claim
 1. 16. Atransformed plant cell having stably incorporated into its genome theconstruct of claim
 2. 17. A method of making a vegetable oil, comprisingthe steps of: a) obtaining the seed of claim 13; and b) extracting oilfrom the seed.
 18. A method of making a vegetable protein, comprisingthe steps of: a) obtaining the seed of claim 13; and c) extracting theprotein from the seed.
 19. A method of directing expression of atranscribable polynucleotide sequence in a plant cell comprisingoperably linking the promoter of claim 1 to the polynucleotide sequenceand transforming a plant cell with the promoter operably linked to thepolynucleotide sequence.
 20. The method of claim 19, comprisingregenerating a plant from the plant cell.
 21. A method of preparing foodor feed comprising a) obtaining the transgenic plant of claim 6; and b)preparing food or feed from said plant or a part thereof.